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-MSH in Obese Women and Their Relationship to Negative Energy Balance
Division of Endocrinology, Department of Internal Medicine (S.-Y.N., K.W.K., K.R.K., S.-K.L.), Yonsei University, College of Medicine, 135-170 Seoul, Korea; Institute of Clinical Chemistry (J.K.), University of Leipzig, 04103 Leipzig, Germany; and Department of Pediatrics, Endocrine Unit (C.M.), Huddinge Hospital, Karolinska Institute, Sweden
Address all correspondence and requests for reprints to: Su Youn Nam, M.D., Ph.D., Division of Endocrinology, Department of Internal Medicine, Yong Dong Severance Hospital, Yonsei University College of Medicine, Young Dong P.O. Box 1217, Seoul, Korea. E-mail: suyoun{at}hotmail.com
Abstract
Leptin and its principal mediators, NPY and 
-MSH are postulated
to play a pivotal role in energy balance. To determine the possibility
of the disturbance in neuropeptides in human obesity and their
consequent changes in response to negative energy balance, we evaluated
plasma and cerebrospinal fluid (CSF) leptin, NPY, and -MSH levels in
obese women before and after weight loss in comparison with normal
control women. Subjects included 16 obese women [mean body mass index
(BMI), 35.6 kg/m2] before and after weight loss induced by
a 2-wk very low caloric diet (800 kcal/d) and 14 normal weight women
(mean BMI, 20.4 kg/m2). The CSF to plasma leptin ratio in
normal weight subjects was 2.3-fold higher than that in obese subjects.
After weight loss in obese subjects, plasma leptin levels decreased by
40% and CSF levels decreased by 51%. There was a positive linear
correlation between CSF and plasma leptin levels at baseline in obese
subjects (r = 0.74, P < 0.05) and a positive
logarithmic correlation in normal weight subjects (r = 0.89,
P < 0.05) and in obese subjects after weight loss
(r = 0.64, P < 0.05). The BMI was negatively
correlated with the CSF to plasma leptin ratio (r = -0.86,
P < 0.05) in all subjects. Neither the baseline
plasma levels nor the baseline CSF levels of NPY were different between
normal weight subjects and obese subjects. After weight loss, the CSF
NPY level decreased significantly compared with baseline values in
obese subjects. The -MSH levels in plasma and CSF did not differ
significantly from controls in obese subjects at baseline or after
weight loss. Baseline CSF leptin level correlated with neither the
baseline CSF NPY level nor the baseline CSF -MSH level.
In conclusion, this study demonstrated that the efficiency of brain
leptin delivery is reduced in human obesity and central nervous system
leptin uptake involves a combination of a saturable and an unsaturable
mechanism. CSF NPY and -MSH did not differ from controls in human
obesity, and the CSF NPY level decreased significantly whereas -MSH
did not differ after weight loss in obese subjects compared with
baseline. There was no significant correlation between CSF leptin and
CSF NPY or -MSH. This could be the result of leptin resistance
present in human obesity and/or the more complex mechanisms involved in
modulating appetite and regulating energy balance in human
obesity.
LEPTIN, ENCODED BY the ob gene, is a 167-amino acid protein produced and released in several tissues in addition to adipocytes (1). It is secreted into the circulation and crosses the blood-brain barrier into the central nervous system (CNS) (2). It acts at the level of the hypothalamus by binding to its receptors and activating secondary signals that inhibit food intake and increase energy expenditure (3). Administration of a small amount of leptin into the cerebral ventricles corrects the obesity and metabolic abnormalities present in the leptin-deficient ob/ob mice (3, 4).
In the great majority of obese humans, however, leptin levels are
increased, indicating that human obesity is leptin resistant
(5). A defect in the blood-brain barrier transport system
to uptake leptin into CNS sites of action has been suggested as the
potential mechanism for this resistance. It is supported by the finding
that the ratio of cerebrospinal fluid (CSF) to plasma leptin decreases
in obese subjects who have a higher plasma leptin level compared with
normal weight individuals (6). Another mechanism for
leptin resistance could be at the level of hypothalamus where the
leptin receptor is heavily expressed (7). As a
result, novel molecular targets and pathways of leptin action have been
identified. Initially among several possible mediators of leptin within
hypothalamus, NPY has been suggested to be an essential conduit for the
leptin signal. It has been demonstrated that leptin directly inhibits
NPY mRNA expression in the arcuate nucleus and, thus, represses the
activity of this potent stimulator of food intake (8, 9, 10).
However, the findings that NPY knockout mice lack feeding or obese
phenotype and that these mice respond normally to the satiety effects
of leptin (11) suggest that other mediators of leptin
exist. In parallel with the NPY pathway, the melanocortin-4 receptor
(MC4-R) has been identified as a downstream effector in the satiety
action of leptin (12). -MSH, derived from POMC, seems
to be an agonist to decrease feeding. In the arcuate nucleus, POMC
neurons express the leptin receptor and POMC mRNA expression is
positively regulated by leptin (13). Thus, it can be
speculated that increased leptin with obesity drives increased arcuate
POMC expression, which then projects -MSH containing axons to MC4-R
expressing cell bodies in the hypothalamus, resulting in decreased food
intake. Considering the above, leptin and its principal mediators, NPY
and -MSH, are postulated to play a pivotal role in energy balance.
The present study investigated plasma and CSF leptin, NPY, and -MSH
levels in normal weight and obese women and the relationship between
CSF leptin and these peptides. Additionally, the consequent changes in
these peptides, in response to negative energy balance, were assessed
in human obesity.
Subjects and Methods
Subjects
Sixteen obese Korean women [mean ± SEM; age, 40.6 ± 2.7 yr; mean body mass index (BMI), 35.6 ± 1.3 kg/m2) and 14 normal weight Korean women (age, 38.8 ± 4.4 yr; BMI, 20.4 ± 2.0 kg/m2) were recruited by local advertisements. None of the subjects were taking any medication or had any evidence of metabolic disease other than obesity and all reported a stable body weight for at least 2 months before the study. Before entering the study, all obese subjects were instructed to maintain a daily diet and routine activities and to record their daily intake and activities during 1 wk in a booklet that was given to them. Their change in body weight during this period was within 1 kg, and their average daily caloric intake was 2000 kcal (range, 1700–2300 kcal/d).
The study was approved by the hospital ethics committee, and informed consent was obtained from each subject.
In all subjects, body composition such as total body fat and lean body mass was determined by a dual-energy x-ray absorptiometer (Lunar DPX; Lunar Corp., Madison, WI).
To study the effect of weight loss, all obese subjects were admitted to the metabolic unit where they stayed for 2 wk maintaining a diet of 800 calories (35% protein, 55% carbohydrates, and 10% fat). Blood and CSF were collected at baseline and on the 15th d from the admission. Ten milliliters of CSF were obtained by lumbar puncture performed over a period of 5 min between 0830 h and 0930 h after an overnight fast and at least 2 h of bed rest. The blood and CSF samples in the normal weight control women were taken at the same time of day and in the same way, as those described for the obese women. There were no complications observed during and after these procedures. CSF was immediately fractionated in several aliquots and stored at -70 C until assayed. Blood sampling was taken 30 min before lumbar puncture, and plasma was immediately separated by centrifugation at 4 C and stored at -70 C until assayed.
Peptide assays
Lepitn was measured in CSF and in plasma by a previously described specific in-house RIA (14). CSF was concentrated by lyophilization and reconstituted in RIA buffer. The sensitivity of the RIA was 0.2 ng/ml. Intra-assay and interassay coefficients of variation were lower than 12.5% in the range between 1–8 ng/ml leptin. Leptin levels of our in-house RIA (x) are comparable with data of a commercially available leptin RIA (y) from Mediagnost (Tuebingen, Germany) in sera of normal weight and obese subjects: y = -0.13 + 0.96x (n = 92: r = 0.94, P < 0.0001).
Plasma and CSF were extracted on disposable reverse phase mini columns
(Sep Column; Peninsula Laboratories, Inc., Belmont, CA)
for NPY and -MSH measurements described by Brunei et al.
(15). The eluted fractions were lyophilized and
reconstituted in RIA buffer. The concentrations in the extracted
samples were measured using a commercially available RIA
(Peninsula Laboratories, Inc.). Sensitivities of NPY and
-MSH assays were 20 pg/tube and 19 pg/tube, respectively. The
corresponding intra-assay coefficients of variations were 1.7% and
2.0%, respectively.
Statistics
The comparison between obese subjects and the normal weight
subjects was performed by unpaired t test. In obese
subjects, comparisons between before and after treatment were analyzed
by paired t test. Correlation was sought by calculating the
Pearson linear r value. Figures 2
and 3 were obtained by using linear
or logarithmic regression. P less than 0.05 was accepted as
the level of significance.
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The characteristics of all subjects are shown in Table 1. In obese subjects, body weight
decreased by 5.3 ± 0.2% (4.79 ± 0.34 kg) of initial body
weight when subjected to a very low calorie diet for 2 wk.
|
), whereas the CSF
leptin levels in obese subjects (0.29± 0.02 ng/ml) were only 2.8-fold
higher than in normal weight subjects (0.10 ± 0.01 ng/ml; Fig. 1B). Consequently the CSF to plasma leptin ratio in normal weight
subjects (mean, 0.03) was 2.3-fold higher than that in obese subjects
(mean, 0.013; Fig. 1C). A 2-wk diet restriction decreased mean body fat
amount by 6.3 ± 0.5% of initial value in obese subjects, and it
was correlated with neither the baseline plasma leptin level nor the
baseline CSF leptin level. After weight loss in obese subjects, the
plasma leptin level (13.0 ± 1.3 ng/ml) decreased by 40% and the
CSF level decreased by 51% (0.15 ± 0.01 ng/ml; Fig. 1, A and B).
The CSF to plasma leptin ratio was slightly lower than the baseline
level (Fig. 1C). There was a positive linear correlation between CSF
and plasma leptin level at baseline in the obese subjects (r =
0.74, P < 0.05) and a positive logarithmic correlation
in normal weight subjects (r = 0.89, P < 0.05)
and in obese subjects after weight loss (r = 0.64,
P < 0.05; Fig. 2
). The
BMI was positively correlated with plasma (r = 0.84,
P < 0.001) and CSF leptin (r = 0.47,
P < 0.05) levels, whereas it was negatively correlated
with the CSF to plasma leptin ratio (r = -0.86, P
< 0.05; Fig. 3) in all subjects. Neither
the baseline plasma levels nor the baseline CSF levels of NPY were
different in normal weight subjects and obese subjects (Fig. 4A). After weight loss the CSF NPY level
decreased significantly in obese subjects compared with baseline values
in obese subjects (Fig. 4A). The -MSH levels in plasma and CSF
did not differ significantly from controls in obese subjects at
baseline or after weight loss (Fig. 4B). Correlation analysis revealed
that baseline CSF leptin level correlated with neither the baseline CSF
NPY level nor the baseline CSF -MSH level.
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This study investigated the concentrations of leptin and its
principal mediators, NPY and -MSH, in CSF and plasma and the
consequent changes in response to negative energy balance in human
obesity. We observed lower CSF to plasma leptin ratios in obese
subjects than in normal weight subjects despite higher leptin
concentrations in plasma and CSF in obese subjects. These findings
support other previous results demonstrating the reduced efficiency of
brain leptin delivery in obese subjects (16, 17). The
association between plasma and CSF leptin concentration was best
described by a logarithmic function in obese subjects after weight
loss, whereas it was a linear relationship at baseline in obese
subjects. These results suggest the possibility of additional leptin
uptake into brain at high plasma leptin level via a nonsaturable
mechanism such as a simple diffusion although substrate saturation
effects on the transporter may be apparent at normal and moderate
plasma leptin levels observed in normal weight and obese subjects after
weight loss. This finding is consistent with other previous reports
describing CSF leptin concentration does not form plateau at high
plasma levels (16, 17). Although this concept has clinical
implications for further increase in CSF leptin when a higher dose of
leptin is administered, additional studies will be needed to evaluate
the efficiency of leptin delivery into the CNS in obese subjects during
high-dose leptin administration.
Among the most promising candidates for leptin-sensitive cells in the hypothalamus are arcuate nucleus neurons that coexpress several neuropeptides mediating the action of leptin on the regulation of energy homeostasis (18, 19). The neurons that produce NPY in the arcuate nucleus of the hypothalamus project to the paraventricular nucleus (10, 20), where they potently stimulate food intake (21, 22). Indeed, underweight anorexia nervosa patients had significantly elevated concentrations of CSF NPY compared with normal controls, and CSF NPY concentrations normalized after long-term recovery of body weight (23, 24). It has also been reported that CSF NPY level was inversely correlated with energy consumption in healthy volunteers (23). Contrary to our expectations based on these findings, the baseline CSF NPY levels in obese subjects were not different from those of normal weight subjects in our study. It is not known why such an expected physiologic relationship was not observed among obese subjects, but prior sustained stable adiposity may uncouple this as a potential mechanism of homeostatic regulation of body weight. Furthermore, considering that leptin itself has the ability to directly suppress hypothalamic NPY synthesis (8), despite the reduction in CSF leptin concentration after weight loss in obese subjects, the significant decrease in CSF NPY levels compared with the baseline values was unexpected. A possible explanation for the apparent discrepancy may be the leptin resistance in human obesity. Chronic increased CSF leptin concentration in obesity could reduce leptin receptor binding or sensitivity on NPY-producing neurons. Alternatively, the sensitivity of NPY gene expression in the arcuate nucleus has been reported to exhibit a body weight-dependent component (25). Djungarian hamsters in a relatively obese state housed during a long photo period did not exhibit a compensatory increase in NPYergic activity when deprived of food, whereas hamsters in a relatively lower body weight state housed during a short photo period exhibited the largest increase in NPY mRNA levels after food deprivation (26). Considering the above, hypothalamic NPY gene expression in obese humans may be relatively insensitive to food deprivation when compared with that in normal or underweight subjects who would be more vulnerable to food deprivation that could be a more immediate survival threat to them. In addition to the orexogenic effect, NPY functions as an endogeneous anxiolytic agent that may buffer against the effects of stress on the CNS (25, 27). In human, increases in circulating NPY levels have been found in response to severe stress conditions (27, 28). In obese subjects, NPY release might be increased with high-intensity stress from diet restriction and hospitalization in the beginning of this study, and then NPY might be depleted by prolonged exposure to stress. This could be another possible explanation for the significant reduction in NPY compared with baseline values in obese subjects after 2-wk diet restriction. In our study, plasma NPY concentrations did not have any relationship with CSF NPY concentrations, which is consistent with other previous studies reporting that NPY concentrations in CSF were very high when compared with peripheral blood levels (15, 29). In the periphery, NPY is present in noradrenergic perivascular, cardiac, enteric and parasympathetic nerves as well as in the adrenal medulla (30). However, the exact nature of the relationship between central and peripheral NPY is poorly understood.
A study with NPY gene knockout mice demonstrated normal patterns of
food intake and body weight (11), which suggests that NPY
secretion in the hypothalamus may not be essential for leptin to
inhibit food intake and that additional factors are involved in
leptin’s control of adiposity. Included among these is -MSH, a
melanocortin cleavage product of POMC. Hypothalamic MC4-R has been
implicated as an essential conduit for the leptin signal in the
regulation of energy balance (18). MC4-R pathway is not
restricted to a single endogenous agonist, -MSH, but also binds to a
competitive antagonist, Agouti-related protein (AGRP)
(31). In the present study, the CSF -MSH level did not
differ significantly from controls in obese subjects at baseline states
or after diet restriction. Interestingly, it has been reported that
diet modulation in rats did not change hypothalamic -MSH and POMC
concentrations but significantly increased AGRP, suggesting that MC4-R
activity may not be regulated by changes in agonist (-MSH) but by
changes in the antagonist (AGRP) availability (32).
Although in humans mutations in the POMC and MC4-R genes result in
phenotypes with profound obesity (33, 34, 35), it remains to
be determined if the hypothalamic MC4-R system operates homeostatically
to attempt to maintain optimal energy stores by modulating activity of
the receptor with tonic -MSH or AGRP release in human. Several
studies have demonstrated that -MSH concentrations in CSF reflect
-MSH secretion within the CNS because the CSF -MSH concentrations
were correlated with -MSH immunoreactivity in various regions of
human brains (36, 37). It has been reported that plasma
-MSH levels do not covary with CSF levels (38). In the
periphery, -MSH is expressed in many kinds of peripheral cells
including phagocytes and keratinocytes (39, 40) and
actions as a potent anti-inflammatory peptide (41, 42). A
significant increase in plasma -MSH concentrations during infection
or inflammatory disorders has been reported (43, 44). At
this time, however, it has not been established whether central and
peripheral -MSH are common systems that responds similarly to
certain stimuli.
In conclusion, this study investigated concentrations of leptin and its
known principal mediators, NPY and -MSH, in the blood and CSF in the
same obese subject and age- and sex-matched normal weight controls and
the consequent change in these peptides in response to negative energy
balance in human obesity. We demonstrated that the efficiency of brain
leptin delivery is reduced in human obesity and that CNS leptin uptake
involves a combination of a saturable and an unsaturable mechanism. CSF
NPY and -MSH did not differ from normal weight controls. CSF NPY
level decreased significantly whereas -MSH did not differ after
weight loss in obese subjects compared with baseline. There was no
significant correlation between CSF leptin and CSF NPY or CSF -MSH.
This could be result of leptin resistance observed in human obesity
and/or more complex mechanisms involved in modulating appetite and
regulating energy balance in human obesity.
Acknowledgments
Footnotes
Abbreviations: AGRP, Agouti-related protein; BMI, body mass index; CNS, central nervous system; CSF, cerebrospinal fluid; MC4-R, melanocortin-4 receptor.
Received October 4, 2000.
Accepted July 5, 2001.
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